The neuronal ceroid lipofuscinoses (NCL)/Batten disorders are a group of closely related hereditary neurodegenerative disorders that affect infants, children and adults, and which occur at a frequency of between 2 and 4 in 100,000 live births. Most forms of NCL afflict children and their early symptoms and disease progression tend to be similar. Initial diagnosis is frequently based upon visual problems (e.g., retinitis pigmentosa), behavioral changes, mental and motor decline and seizures (1). Progression is reflected by a decline in mental abilities, increasingly severe and untreatable seizures, blindness and loss of motor skills while further progression can result in dementia or a vegetative state. The pathologic hallmark of NCL is neuronal loss. Theories pertaining to pathophysiology include increased lipid peroxidation, alterations in dolichol turnover, increased inflammatory responses, unexplained accumulation and processing of subunit c of mitochondrial ATP synthase, and accelerated apoptosis (2, 3). There is no effective treatment for NCL and all childhood forms are eventually fatal. Several forms of NCL are differentiated according to age of onset, clinical pathology and genetic linkage. These include early infantile NCL (INCL, CLN1), late infantile NCL (LINCL, CLN2), juvenile NCL (JNCL, CLN3) adult NCL (CLN4), two variant forms of LINCL (CLN5 and CLN6) and possibly other atypical forms.
Juvenile neuronal ceroid lipofuscinosis (JNCL), the most common form of NCL, is due to mutations in the CLN3 gene (4). More than 35 mutations are known, most due to a 1.02 kb deletion in genomic DNA, which produces a premature stop codon and loss of nucleotides 461-677. This results in a truncated protein 181 amino acids in length. The CLN3 protein (CLN3p) has four potential glycosylation sites, 12 phosphorylation sites and a farnesylation site. The CLN3 protein imparts anti-apoptotic properties to cells and neurons. Two conserved amino acid stretches within exons 11 and 13, and two of the CLN3p glycosylation sites are necessary for preservation of this function (5). Membrane topology studies suggest that the CLN3p has five transmembrane domains with an extracellular/intraluminal amino-terminus and a cytoplasmic carboxy terminus (6).
CLN3p is highly conserved in eukaryotes and is ubiquitously expressed in mammals. CLN3p imparts anti-apoptotic properties to neurons and other cells (7-9) and regulates autophagy (10). CLN3-deficient cells grow slowly, have enhanced sensitivity to apoptosis and altered levels of ceramide and sphingomyelin. These deficits can be corrected following restoration of CLN3p to CLN3-deficient cells. Additionally, CLN3 mRNA and protein levels are over-expressed in cancer cells (12).
A comprehensive localization study has demonstrated that wild type CLN3p is localized to Golgi apparatus and plasma membranes and traffics via early recycling RAB4- and Rab11-positive endosomes from the Golgi apparatus to lipid rafts (LR) (20). This was established in primary rat hippocampal neurons, post-mitotic human neurons and normal human fibroblasts. Mutant CLN3p localizes to a disrupted Golgi apparatus, fails to reach the plasma membrane and partially mis-localizes to lysosomes.
CLN3p harbors a conserved motif, 291VYFAE295, necessary for its impact on cell growth and apoptosis (20). This motif is embedded in a stretch of amino acids structurally homologous to a GalCer lipid raft binding domain. This domain (21) defines a lipid raft binding site that is structurally identical to the one in prionic protein, PrP and the V3 loop of the HIV-1 surface envelope glycoprotein, gp120. Dual immuno-labeling studies localized wild-type CLN3p with alkaline phosphatase and caveolin-1 to lipid rafts and caveolae in some cell types (20). There was minimal co-localization of mutant CLN3p with these lipid raft markers.
Lipid rafts are involved in multiple cellular processes, including protein trafficking, signaling complex formation and signal transduction events pertinent to apoptosis, cell adhesion (22), stress responses, regulation of the cytoskeleton, conduction of proathrogenic stimuli and immune cell function (21-24). They also serve as portals of entry for toxins, viruses and bacteria (26, 29, 30). Additionally, lipid rafts are important for normal synapse density and morphology in the central nervous system (31), myelin integrity and myelin-axonal interactions (21). Lipid rafts are liquid-ordered microdomains of plasma membrane that are insoluble in non-ionic detergents. These domains are thought to derive from the Golgi apparatus and are made up of glycosphingolipids and cholesterol and are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins (32). They also harbor the sphingolipid, ceramide, a pro-apoptotic lipid second messenger (33, 34). Protein prenylation promotes association of proteins to lipid rafts and CLN3p is prenylated (35). Lipid rafts house caspase-8, the first initiator caspase to be activated in the apoptotic cascade in CLN3-deficient cells (36). Morphologically, in CLN3p-deficient cells, raft vesicular structures are small compared to those derived from normal cells as demonstrated by transmission electron microscopy (TEM). These structural differences may reflect altered sphingolipid composition of CLN3-deficient lipid rafts.
The present invention overcomes previous shortcomings in the art by providing methods and compositions to treat disorders associated with a deficiency in a gene product of a CLN gene (e.g., CLN1, CLN2, CLN3, CLN5, CLN6, CLN7, CLN8, CLN9 or CLN10/CTSD and/or CLCN6).